This invention relates to polyethylene glycol derivatives which are novel and of the use as peptide (particularly protein)-modifying reagents, peptides having guanidino group which are modified by said polyethylene glycol derivatives and the methods for producing them.
In recent years, with the development of research in proteins, a great number of peptides, in particular, physiologically active proteins having various actions have been found. Owing to the progress of genetic recombination techniques and organic synthetic methods of peptides, it has become possible to obtain these physiologically active peptides and their structurally analogous compounds in a large amount. Many of these peptides having special activity are extremely useful as pharmaceuticals.
However, it is known that the clearance of peptides which has been administered in the circulatory system is generally very fast, and therefore the improvement in durability of such peptides has been desired. Besides, since there is a risk of causing serious symptoms due to the production of antibodies in the case where the peptides are obtained from different species of animals or designed by peptide-protein engineering, and they are different from those from humans in structure, the improvement of the antigenicity of said proteins is desired.
In order to use these peptides as pharmaceuticals, it is necessary to solve said problems in the aspect of their antigenicity and durability. The method of modifying the peptides chemically with macromolecular compounds is known to be extremely effective as the means by which to solve the above-mentioned problems.
Thus, polyethylene glycol derivatives have been widely used as peptide-modifying macromolecular reagents because they have excellent characteristics that they do not have immunogenicity themselves and that they do not affect the three-dimensional structures of peptides (proteins) in aqueous solutions.
In modifying peptides with polyethylene glycol derivatives, there have been known, as the general methods for activation, the activation method with triazine derivatives [Inada et al, Jpn. J. Cancer Res. (Gann), 77, 1264-1270 (1986)], the active ester method with N-hydroxysuccinimide [Leonardo M. et al, Tetrahedron, 40, 1581-1584 (1984)], the activation method with carbonyldiimidazole [Charles. O. Beecham et al, Anal. Biochem, 131, 25-33 (1983)], the activation method with aldehydes [Fujino et al, Japanese Patent Application Unexamined Publication (Kokai) No. 178926/1986] and so on.
Any of these modification methods, however, comprise modifying the amino groups at the N-terminal or in the side chain of the lysine residues of the peptides.
On the other hand, in many peptides, the amino groups at the N-terminal or those in the side chain of the lysine play an important role for the expression of physiological activities. Therefore, in the case of these peptides, modifying the amino groups with the above-mentioned activating reagents is not preferable because such modification results in the reduction of activities. Besides, in the case of the method in which the lysine residues alone are modified, there is a limitation on the sites of modification. As it is considered to be effective for the propose of controlling properties of peptides to modify various sites other than the amino groups, extremely significant is the development of reagents modifying the other functional groups.
As the reagents modifying the other functional groups hitherto known, mention is made of maleimide derivatives modifying the mercapto group and amino derivatives modifying the carboxyl group [Frank F. Davis et al, Japanese Patent Application Examined Publication (Kokoku) No. 23587/1981] and so on.
However while, unless mercapto groups exist in the form of mercapto groups on the surface of the molecules, the modification thereof is considered to be difficult, and there are a very few peptides having such structures.
In addition, in the case of modification with amino derivatives, it is difficult to react the desired modifying reagent alone selectively since the side reactions by the amino groups in the peptides occur.
On the other hand, as the lower molecular reagents for modifying guadino groups in peptides, there are known phenylglyoxal (Journal of Biological Chemistry, vol. 248, 6171 (1968)), 2,3-butanedione (Biochemistry, vol. 12, 3915 (1973)), 1,2-cyclohexanedione (Journal of Biological Chemistry, vol. 250, 557 (1975)) and so on. However, there have not been so far known any polyethylene glycol derivatives capable of modifying guanidino groups in peptides.